The genetic modification of T lymphocytes is the basis for novel approaches to study and establish tumor immunity. The genetic transfer of antigen receptors is indeed a powerful approach to rapidly generate tumorspecific T lymphocytes. However, while necessary, tumor antigen recognition is not sufficient to permit tumor eradication. To achieve this goal, primed CTLs must expand to sufficient numbers, migrate to tumor sites, mature into effector cells and carry out their cytolytic function unimpeded. The genetic strategies we are pursuing aim to increase recognition of tumor antigens, enhance anti-tumor functions and sustain T cell function in cancer patients. Most important for our understanding of human tumor immunology and therapeutic goals, gene addition and knockdown strategies can be applied to human T lymphocytes, on which we focus in this project. The specific aims are based on our published and preliminary data. Aim 1: To investigate the biological properties and therapeutic potential of tumor specific CD8+ T cells co-stimulated by CD28 and 4-1BB. We hypothesize that concerted CD28 and 4-1BB signals sustain CD8+ T cell proliferation and survival, and may thus augment the therapeutic potency of genetically targeted T lymphocytes. Aim 2: To investigate the effect of ex vivo IL-15 on the proliferation, survival, differentiation and therapeutic potential of adoptively transferred human primary T lymphocytes. This aim builds on our recent finding that IL-15 enhances the therapeutic potential of cultured 19z1-transduced PBLs upon adoptive transfer to tumorbearing mice. We hypothesize that IL15 increases therapeutic efficacy through several mechanisms, most importantly T cell survival. Aim 3: To establish an efficient protocol for T'cell transduction and expansion in a closed system and perform a phase I clinical trial to assess the safety, persistence and therapeutic activity of autologous 19-28z-transduced T lymphocytes in patients with relapsed, chemo-refractory chronic lymphocytic leukemia. We hypothesize that patient T cells expressing a CD28/ -like chimeric antigen receptor will induce durable remissions, more so in cyclophosphamide-treated recipients. Our investigation critically depends on the Gene Transfer and Somatic Cell Engineering Core, to genetically modify patient T cells, the Imaging Core, to study T cell migration and persistence in vivo, and the Administrative Core, for biostatistics and data management.